In vivo genome correction in a hemophilia mouse model
Conventional gene therapy inserts the wildtype, or normal, gene into the genome. While this allows for the correct gene to be expressed in the cell, often times, the gene gets introduced to parts of the genome that eventually prevents endogenous expression of essential genes. Genomic integrity is compromised, and the result could be detrimental. In order to combat this potentially devastating consequence, a more targeted approach has been taken. One of the ways in which gene therapy focuses its effects would be to use a cell-type specific promoter- if the target gene is also cell-type specific. But this still does not address the issue of off-site insertions. A new strategy of using the cells' innate chromosome repair machinery has been adapted to insert the correct gene in vivo. Hemophilia Hemophilia patients (Hemophilia A or B) lack the coagulation factors that help propagate the clotting cascade. Under normal conditions, these clotting factors congregate at the site of the injury to ultimately generate thrombin, which is the serine protease responsible for creating the fibrin meshwork that physically forms the plug on the vascular wall. Hemophilia A patients are missing the clotting factor VIII, a cofactor for factor IX- the enzyme that activates another serine protease Factor X. Hemophilia B is a genetic disorder in which the affected person does not express any active coagulation factor IX. Either this is functionally null, or truncated/misfolded protein. Affected individuals express less than 1% of normal level of FIX, which is the serine protease that activates FX, the main enzyme that generates thrombin, a hemostatic enzyme that cleaves fibrinogen to fibrin that creates the meshwork that physically stops the bleeding. Hemophilia as a candidate for gene therapy Hemophilia is an attractive target for gene correction therapy, as it is a monogenic disorder, and only require about 5% percent of normal expression of the protein to be within the therapeutic range. Furthermore, the expression is exclusiely in the liver- the only organ that expresses the clotting factors. In vivo gene correction using zinc finger nucleases Zinc fingers can recognize specific sequences on DNA strands. An endonuclease has been attached to a ainc nuclease which enhances the fidelity of the cleavage site. If zinc finger nucleases that recognize sequences upstream of the Fok1 cleavage site on the complementary strands, then each endonucleases can cut the strand, causing a double stranded break. One of the more common ways of double stranded break repair mechanisms is non-homologous end joining (NHEJ). By creating this double stranded break, the DNA repair pathway is stimulated, and enhances the chance that the correct, wild-type gene is incorporated in the repair process, through homologous recombination. Adverse effects Aside from off-target inclusion of the wildtype gene, which can induce tumorigenesis, gene therapy with viral vectors can induce an immune response that will decrease the expression of the therapeutic expression of the coagulation factor. One of the subjects were shown to have immune responses to the vector dose2 a few weeks after administration of the viral vector. This adverse effect was ameliorated with the profilactic administration of the steroid prednisolone before immunogenecity. This is most likely due to an immune response to the viral capsid protein on AAV8 vector used for therapeutic expression of the missing factor. References Li H, et al. (2011) In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 475(7355):217-221. Nathwani AC, et al. (2011) Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. The New England journal of medicine 365(25):2357-2365.